Khat, (Catha edulis Forssk., Celastraceae) is an evergreen tree indigenous to East Africa and Yemen. The fresh young leaves are commonly chewed, known as the Khat habit, to alleviate hunger and to produce stimulating effects (CNS). Such effects were shown to be due to phenylalkylamine alkaloids, primarily cathinone (Kalix 1990; Crombie et al. 1990). Habitual use of khat is often associated with social and medical problems (Shadan and Shellard 1972).

Previous work on khat cultures, in our laboratories, has dealt with in vitro micropropagation (Elhag 1991) and the production of secondary metabolites by micropropagated plantlets and callus cultures (El-Domiaty et al. 1994). In the course of our work with khat tissue cultures, the production of dark colored pigments was observed as a typical characteristic of the callus culture (Elhag and Mossa 1996). The present investigation deals with the isolation, identification, and biological effects of such pigments.

METHODOLOGY

Callus Culture

Leaves from young twigs of micropropagated greenhouse grown plants (Elhag 1991) were used as explants for callus induction and as fresh material for extraction and chemical analysis. As described previously by El-Domiaty et al. (1994) callus induction from leaf sections was best achieved on MSB5 basal medium (Murashige and Skoog inorganic salts with Gamborg-B5 vitamins) supplemented with 3.0 mg/L of either IBA (indolebutyric acid) or NAA (naphtalene acetic acid). PVP (polyvinylpyrrolidone) at a concentration of 0.1% in the medium was found beneficial in enhancing callus growth. However, PVP was excluded from the culture medium after the establishment of proliferating cultures. Callus tissues collected form several subcultures on IBA or NAA-containing media were used for extraction and analysis of pigments.

Extraction and Isolation

The freeze-dried powdered callus of C. edulis (25 g) was extracted with MeOH at room temperature. The MeOH extract was evaporated and the residue (3.9 g) was partitioned between CHCl3 and H2O. The dark brown residue (0.89 g) left after evaporation of CHCl3 was chromatographed on silica gel column (2 × 20 cm) and eluted with CHCl3 (300 ml) and CHCl3 containing a trace of acetic acid (10:0.01, 200 ml). Two fractions were collected, fraction A (230 mg) and fraction B (110 mg). Fraction A (containing the pigments) was further chromatographed on reversed phase silica gel (C-18, 2540 m) using medium pressure column (2 × 18 cm, 75% MeOH in 5% aqueous acetic acid) with a flow rate of 3 ml/min. Upon evaporation, fractions eluted between 2560 ml gave 32 mg of compound 1 (0.13%), while fractions 120175 ml gave 120 mg of compound 2 (0.48%).

Antimicrobial Assays

Evaluation of the antimicrobial activity of callus extracts (petroleum ether, CHCl3, MeOH, and aqueous successive extracts) and the isolated compounds 1 and 2 was conducted according to the disk diffusion and agar dilution methods (Mitscher et al. 1972; Jayasuria 1988). Chloramphenicol and streptomycin were used as positive controls. The solvent DMSO was used as the negative control for all the experiments. The bacteria used were obtained from the National Collection of Type Culture (NCTC), Central Public Health Laboratory, London; and the Center for Disease Control, Atlanta, Georgia, US. The minimum inhibitory concentration (MIC) of the two compounds (22b-hydroxytingenone and tingenone) was determined by the two-fold serial dilution assay (Hufford et al. 1975). The MIC was taken as the lowest concentration that inhibited growth after 48h of incubation at 37°C for B. subtilis, S. aureus, and S. durans and after 72 hr for Mycobacterium strains.

Cytotoxicity and Anti-HIV

The assays were performed according to the standard procedures of the in vitro primary screen of NCI (Weislow et al. 1989; Grever et al. 1992).

EXPERIMENTAL RESULTS

Extract Identification

Khat callus produced on MSB5 medium containing IBA or NAA continued to proliferate as compact hard clumps with snow-white top surfaces and dark-pigmented lower parts. The dark pigments partly diffused into the medium at the point of contact with the agar medium. This dark pigmentation was thought to result from the high content of polyphenols (tannins) that is known for the intact plant (El-Sissi and Abdalla 1966). However, the initial TLC (Si gel, EtOAc-HCOOH-H2O, 90:6:5) screening of the EtOH extract of the callus detected only a trace of the polyphenolic precursors of tannins gallocatechin and epigallocatechin. On the other hand, intact khat plants did not show the same pigments detected in the callus (TLC : Si gel, EtOAc-HCOOH-toluene, 9:1:10).

Compound 2 gave [a]D-307.4°; its UV spectrum [lmax) MeOH : 420, 286 and 252 nm], was quite similar to that of compound 1, suggesting the presence of a similar chromophore, IR spectrum of compound 2 showed the same characterstic bands of 1. 1H- and 13C-NMR data of compound 2 were similar to that of 1 except that 2 showed the absence of hydroxyl group at C-22, as it appeared at d 52.6 in 13C-NMR and one of the H-22 protons appeared at d 2.92 (H, d, J= 14 Hz) in 1H-NMR. Compound 2 was thus identified as tingenone; this was confirmed by comparison with reported data (Gonzalez et al. 1975; Kutney et al. 1981; Ngassapa et al. 1994).

Antimicrobial Activity

Initial antimicrobial screening of the crude callus extracts was conducted using the disk diffusion method and was confirmed with bioautography (Jayasuria 1988). The highest growth inhibition was found in the petroleum ether and chloroformic successive extracts, the latter being more active. Further fractionation and purification of the chloroformic-soluble fraction of the methanolic extract (as described in Materials and Methods), resulted in the isolation of two active compounds (1) and (2). The isolated compounds were identified as 22b-hydroxytingenone (1) and tingenone (2) by various spectral methods. Both compounds exhibited significant activities against B. subtilis, S. aureus, and S. durans (MIC 0.6 mg/ml), being more potent than the positive control chloramphenicol (Table 1). Both compounds were also found to be more potent against Mycobacterium species (MIC was 5.0 mg/ml for both compounds) than the positive controls, streptomycin and isonicotinic acid hydrazide (Table 1). However, both compounds; were found to be inactive against the gram-negative bacteria E Coli and the fungus C. albicans (Table 1).

Table 2. Results of antitumor evaluation of 22b-hydroxy-tingenone in the NCI in vitro primary screen.

Panel

ED50 (mg/ml)

Leukemia

0.54

Non-small cell lung cancer

4.4

Colon cancer

1.31

CNS cancer

3.3

Melonama

1.71

Ovarian cancer

2.35

Renal cancer

1.61

Prostate cancer

0.85

Breast cancer

1.11

CONCLUSIONS

The cultural conditions for khat callus induction and growth were established. Best callus induction and growth occurred on MSB5 medium supplemented with 3.0 mg/L of either NAA or IBA. The production of dark pigments was observed at the start of callus induction and continued with subcultures as a typical characteristic of khat callus.

The isolation of 22b-hydroxytingenone and tingenone from khat callus cultures is reported for the first time. They could not be detected in the mother plant grown in the greenhouse. The crude callus extracts and the isolated compounds, 22b-hydroxytingenone (compound 1) and tingenone (compound 2), showed high antibacterial activity against gram positive and myocbacteria and broad cytotoxic activity against several cell-line systems.

Large scale production of khat cultures for the commercial production of such biologically active components is a promising system worthy of further investigation. It would be ironic if khat, which is considered a plant of abuse, turned out to be a miracle plant with efficacious medical properties.